The proposal presented here seeks to identify small RNA-dependent heterochromatin loci in the mammalian genome. The potential for small RNAs to direct heterochromatin has been suggested in other species. There have been suggestions that such heterochromatin exists in mammals, but evidence of this type of silencing occurring at endogenous loci has remained elusive. The finding of such a mechanism could have profound impacts on both mammalian development and disease. It would uncover new roles for small RNAs in mammalian cells, which would require a revaluation of the roles of small RNAs and how those might be involved in heterochromatin formation during the course of normal development. Additionally, these findings could suggest new mechanisms for how alterations in the heterochromatin landscape of cancers could occur. Finally, looking ahead, a complete understanding of how small RNAs can direct heterochromatin may allow the generation of therapies that would specifically silence detrimental genes in a variety of disease states. To identify potential regions of the mouse genome where heterochromatin is dependent on small RNAs, I will first examine the heterochromatin landscape in mouse ES cells that are wild type, or deficient in small RNAs. I will take advantage of the next-generation ChlP-sequencing technology to survey the entire genome in an unbiased manner. A preliminary analysis of H3K9me3-associated heterochromatin has already revealed one such locus. Using a directed approach, I will examine the dependency of other chromatin marks on small RNAs. Next, I will elucidate the proteins necessary for small RNA-dependent heterochromatin. To accomplish this, I will use a combination of knockdowns and ChIP to examine the proteins that are present at this locus (or loci) and are necessary for heterochromatin. I will also examine if exogenously added small RNAs can re-establish heterochromatin in the absence of Dicer. Finally, I will expand my analysis to differentiated tissues. I will use mouse embryonic fibroblasts (MEFs) to compare the small RNA populations and heterochromatin landscape between wild type MEFs and MEFs deficient in producing small RNAs. Public Health Relevance: The cohort of genes that are "on" and "off' define the numerous cell types in our bodies, and are often misregulated in diseases such as cancer. Two mechanisms for turning genes off have been observed to interact in non-mammalian eukaryotes, but this link has been elusive in mammals. Identifying this link could reveal new paradigms for switching genes from "on" to "off' and may suggest new therapies.